Follistatin-like protein 1 is a critical mediator of experimental Lyme arthritis and the humoral response to Borrelia burgdorferi infection

Abstract

Follistatin-like protein 1 (FSTL-1) has recently been described as a critical mediator of CIA and a marker of disease activity. Lyme arthritis, caused by Borrelia burgdorferi, shares similarities with autoimmune arthritis and the experimental murine model collagen-induced arthritis (CIA). Because FSTL-1 is important in CIA and autoimmune arthritides, and Lyme arthritis shares similarities with CIA, we hypothesized that FSTL-1 may be an important mediator of Lyme arthritis. We demonstrate for the first time that FSTL-1 is induced by B. burgdorferi infection and is required for the development of Lyme arthritis in a murine model, utilizing a gene insertion to generate FSTL-1 hypomorphic mice. Using qPCR and qRT-PCR, we found that despite similar early infectious burden, FSTL-1 hypomorphic mice have improved spirochetal clearance in the face of attenuated arthritis and inflammatory cytokine production. Further, FSTL-1 mediates pathogen-specific antibody production and antigen recognition when assessed by ELISA and one- and two-dimensional immunoblotting. This study is the first to describe a role for FSTL-1 in the development of Lyme arthritis and anti-Borrelia response, and the first to demonstrate a role for FSTL-1 in response to infection, highlighting the potential for FSTL-1 as a target in the treatment of B. burgdorferi infection.

Keywords: Arthritis; Borrelia burgdorferi; FSTL-1; Lyme disease; Mouse model; humoral immunity.

Fig. 1

FSTL-1 hypomorphic mice have decreased fstl-1 gene transcription and FSTL-1 serum protein. (A). A 2565 bp insert containing loxP sequence was inserted within the fstl-1 exon 1 along with flP, a neomycin cassette, a second flP and second loxP site, and was (B) detectable by PCR as FSTL-1 hypomorphic mice yielded a 238 bp gene product (hypo) while wild-type (WT) mice yielded a 199 bp band; in heterozygotes (het) both gene products were detectable. (C) This insertion resulted in FSTL-1 hypomorphic mice with a 60% reduction in serum FSTL-1 (D). Hypomorphic mice showed a reduction in joint fstl-1 transcription versus wild-type mice at day 14 and 42 post infection. Data are representative of at least two individual experiments with n = 10 mice/treatment group. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2

FSTL-1 mediates the development of Lyme arthritis. (A) DBA/1WT (squares) and FSTL-1 hypomorph mice (circles) were infected with 10⁶ Borrelia burgdorferi (solid) or media alone (open). (B) H&E stained tibiotarsal joints of day 42 Borrelia burgdorferi-infected WT and FSTL-1 hypomorph mice were scored for inflammation. Shown are representative 4× (C, E) and 10× (D, F) images of day 42 Bb infected WT (C, D) and FSTL-1 hypomorphic (E, F) mice. Arrow denotes synovial proliferation. b, Bone; s, synovium. *p = 0.03; **p < 0.01. Data are representative of at least two individual experiments with n = 10 mice/treatment group.

Fig. 3

FSTL-1 hypomorphic mice have attenuated Borrelia burgdorferi burden in target tissues. qPCR for ospC of extracted DNA from (A) hearts and (B) joints of WT and FSTL-1 hypomorph mice 14 and 42 days post infection with 10⁶ Borrelia burgdorferi. Error bars represent SEM. Data are representative of at least two individual experiments with n = 10 mice/treatment group. **p < 0.01; ***p < 0.001.

Fig. 4

FSTL-1 influences inflammatory cytokine production in vivo. WT and FSTL-1 hypomorph mice were infected with 10⁶ Borrelia burgdorferi and qRT-PCR performed on (A) hearts and (B) joints for IL-6, IL-1β and TNFα at indicated time points. n = 10 mice/treatment group. Each dot represents an individual animal tissue. Bars represent mean for each treatment group. *p < 0.05.

Fig. 5

FSTL-1 influences immunoglobulin production in response to B. burgdorferi infection. ELISA plates coated with B. burgdorferi membrane-associated proteins were probed with individual B. burgdorferi-infected DBA/1 WT and FSTL-1 hypomorphic mouse serum and detected using class- and isotype-specific secondary antibodies at (A) 14 days post infection and (B) 42 days post infection. Data are representative of at least two individual experiments with n = 10 mice/treatment group. Each ELISA was performed ≥3 times. Mean values are depicted with standard error.

Fig. 6

FSTL-1 is critical to Borrelia burgdorferi antigen recognition. (A) B. burgdorferi membrane-associated proteins (MAP) (15 μg) separated by one-dimensional SDS–PAGE were probed with pooled serum (n = 10 per group) from WT and FSTL-1 hypomorphic mice at 42 days post B. burgdorferi infection with detection of polyclonal IgG/IgM reactivity. Fifty micrograms of B. burgdorferi MAP were separated using IPG followed by SDS–PAGE and transferred to nitrocellulose membrane. Pooled sera from both (B) WT and (C) FSTL-1 hypomorphic mice (n = 10) were used to probe blots followed by detection of polyclonal IgG/IgM reactivity. Resulting two-dimensional serologic maps were correlated with previously identified proteins. Bold and underlined proteins indicate MAPs recognized by WT but not FSTL-1 hypomorphic serum. Data are representative of two individual experiments with n = 10 mice/treatment group.